Human Pluripotent Stem-Cell-Derived Cortical Neurons Integrate Functionally into the Lesioned Adult Murine Visual Cortex in an Area-Specific Way

The transplantation of pluripotent stem-cell-derived neurons constitutes a promising avenue for the treatment of several brain diseases. However, their potential for the repair of the cerebral cortex remains unclear, given its complexity and neuronal diversity. Here, we show that human visual cortical cells differentiated from embryonic stem cells can be transplanted and can integrate successfully into the lesioned mouse adult visual cortex. The transplanted human neurons expressed the appropriate repertoire of markers of six cortical layers, projected axons to specific visual cortical targets, and were synaptically active within the adult brain. Moreover, transplant maturation and integration were much less efficient following transplantation into the lesioned motor cortex, as previously observed for transplanted mouse cortical neurons. These data constitute an important milestone for the potential use of human PSC-derived cortical cells for the reassembly of cortical circuits and emphasize the importance of cortical areal identity for successful transplantation. Espuny-Camacho et al. show that transplanted ESC-derived human cortical neurons integrate functionally into the lesioned adult mouse brain. Transplanted neurons display visual cortical identity and show specific restoration of damaged cortical pathways following transplantation into the visual but not the motor cortex, suggesting the importance of areal-identity match for successful cortical repair

Mixed-species RNA-seq for elucidating non-cell-autonomous control of gene transcription

Transcriptomic changes induced in one cell type by another mediate many biological processes in the brain and elsewhere; however, achieving artefact-free physical separation of cell types to study them is challenging and generally only allows for analysis of a single cell type. We describe an approach employing co-culture of distinct cell-types from different species, which enables physical cell sorting to be replaced by in silico RNA sequencing (RNA-seq) read sorting due to evolutionary divergence of mRNA sequence. As an exemplary experiment, we describe the co-culture of purified neurons, astrocytes, and microglia from different species (12–14 days). Following conventional RNA-seq, we then describe how to use our Python tool Sargasso (http://statbio.github.io/Sargasso/) to separate reads according to species and how to eliminate any artefacts borne out of imperfect genome annotation (10 hours). We show how this procedure, which requires no special skills beyond those that might normally be expected of wet-lab and bioinformatics researchers, enables the simultaneous transcriptomic profiling of different cell types, revealing the distinct influence of microglia on astrocytic and neuronal transcriptomes under inflammatory conditions

Physiological normoxia and absence of EGF is required for the long-term propagation of anterior neural precursors from human pluripotent cells

Widespread use of human pluripotent stem cells (hPSCs) to study neuronal physiology and function is hindered by the ongoing need for specialist expertise in converting hPSCs to neural precursor cells (NPCs). Here, we describe a new methodology to generate cryo-preservable hPSC-derived NPCs that retain an anterior identity and are propagatable long-term prior to terminal differentiation, thus abrogating regular de novo neuralization. Key to achieving passagable NPCs without loss of identity is the combination of both absence of EGF and propagation in physiological levels (3%) of O2. NPCs generated in this way display a stable long-term anterior forebrain identity and importantly retain developmental competence to patterning signals. Moreover, compared to NPCs maintained at ambient O2 (21%), they exhibit enhanced uniformity and speed of functional maturation, yielding both deep and upper layer cortical excitatory neurons. These neurons display multiple attributes including the capability to form functional synapses and undergo activity-dependent gene regulation. The platform described achieves long-term maintenance of anterior neural precursors that can give rise to forebrain neurones in abundance, enabling standardised functional studies of neural stem cell maintenance, lineage choice and neuronal functional maturation for neurodevelopmental research and disease-modelling

Peroxisome Proliferator-Activated Receptor Gamma Enhances the Activity of an Insulin Degrading Enzyme-Like Metalloprotease for Amyloid-β

peer reviewedPeroxisome proliferator-activated receptor gamma (PPARgamma) activation results in an increased rate of amyloid-beta (Abeta) clearance from the media of diverse cells in culture, including primary neurons and glial cells. Here, we further investigate the mechanism for Abeta clearance and found that PPARgamma activation modulates a cell surface metalloprotease that can be inhibited by metalloprotease inhibitors, like EDTA and phenanthroline, and also by the peptide hormones insulin and glucagon. The metalloprotease profile of the Abeta-degrading mechanism is surprisingly similar to insulin-degrading enzyme (IDE). This mechanism is maintained in hippocampal and glia primary cultures from IDE loss-of-function mice. We conclude that PPARgamma activates an IDE-like Abeta degrading activity. Our work suggests a drugable pathway that can clear Abeta peptide from the brain

Brain regional identity and cell type specificity landscape of human cortical organoid models

ABSTRACTIn vitro models of corticogenesis using mouse and human pluripotent stem cells (PSC) have greatly improved our understanding of human brain development and disease. Among these, 3D cortical organoid systems are able to recapitulate some aspects of in vivo cytoarchitecture of the developing cortex.Here, we tested three cortical organoid protocols for brain regional identity, cell type-specificity and neuronal maturation. Overall all protocols gave rise to organoids that displayed a time-dependent expression of neuronal maturation genes such as those involved in the establishment of synapses and neuronal function. We showed that three months old cortical organoids showed a pattern of gene expression that resembled late human embryonic cortex. Comparatively, directed differentiation methods without WNT activation gave rise to the highest degree of cortical regional identity in brain organoids. Whereas, default “intrinsic” brain organoid differentiation produced the broadest range of cell types such as neurons, astrocytes and hematopoietic-lineage derived microglia cells of the brain. These results suggest that cortical organoid models produce diverse outcomes in terms of brain regional identity and cell type specificity and emphasize the importance of selecting the correct model for the right application.</jats:p

Restoration of Progranulin Expression Rescues Cortical Neuron Generation in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia

SummaryTo understand how haploinsufficiency of progranulin (PGRN) causes frontotemporal dementia (FTD), we created induced pluripotent stem cells (iPSCs) from patients carrying the GRNIVS1+5G > C mutation (FTD-iPSCs). FTD-iPSCs were fated to cortical neurons, the cells most affected in FTD. Although generation of neuroprogenitors was unaffected, their further differentiation into CTIP2-, FOXP2-, or TBR1-TUJ1 double-positive cortical neurons, but not motorneurons, was significantly decreased in FTD-neural progeny. Zinc finger nuclease-mediated introduction of GRN cDNA into the AAVS1 locus corrected defects in cortical neurogenesis, demonstrating that PGRN haploinsufficiency causes inefficient cortical neuron generation. RNA sequencing analysis confirmed reversal of the altered gene expression profile following genetic correction. We identified the Wnt signaling pathway as one of the top defective pathways in FTD-iPSC-derived neurons, which was reversed following genetic correction. Differentiation of FTD-iPSCs in the presence of a WNT inhibitor mitigated defective corticogenesis. Therefore, we demonstrate that PGRN haploinsufficiency hampers corticogenesis in vitro

Altered neuronal network and rescue in a human MECP2 duplication model

Submitted by Nuzia Santos ([email protected]) on 2016-07-13T18:33:59Z No. of bitstreams: 1 ve_Nageshappa_Savitha_Altered_CPqRR_2016.pdf: 973558 bytes, checksum: 36ce1b9b175e435858d7c9a9c623198a (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-07-13T18:45:46Z (GMT) No. of bitstreams: 1 ve_Nageshappa_Savitha_Altered_CPqRR_2016.pdf: 973558 bytes, checksum: 36ce1b9b175e435858d7c9a9c623198a (MD5)Made available in DSpace on 2016-07-13T18:45:47Z (GMT). No. of bitstreams: 1 ve_Nageshappa_Savitha_Altered_CPqRR_2016.pdf: 973558 bytes, checksum: 36ce1b9b175e435858d7c9a9c623198a (MD5) Previous issue date: 2016Center for Human Genetics. Laboratory for the Genetics of Cognition. KU Leuven, BelgiumUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAUniversité Libre de Bruxelles. Institut de Recherches en Biologie Humaine et Moléculaire.Brussels, Belgium/ VIB Center for the Biology of Disease. Leuven, BelgiumVIB Center for the Biology of Disease. Leuven, Belgium/KU Leuven Center for Human Genetics and Leuven Institute for Neurodegenerative Diseases. KULeuven, BelgiumUniversité Libre de Bruxelles. Institut de Recherches en Biologie Humaine et Moléculaire.Brussels, Belgium/ VIB Center for the Biology of Disease. Leuven, Belgium/ WELBIO. Brussels, BelgiumDepartment of Development and Regeneration. Stem Cell Institute Leuven. KU Leuven Medical School. Cluster Stem Cell Biology and Embryology. Leuven, BelgiumDepartment of Development and Regeneration. Stem Cell Institute Leuven. KU Leuven Medical School. Cluster Stem Cell Biology and Embryology. Leuven, BelgiumDepartment of Development and Regeneration. Stem Cell Institute Leuven. KU Leuven Medical School. Cluster Stem Cell Biology and Embryology. Leuven, Belgium/Manipal Institute of Regenerative Medicine. Bangalore, IndiaBaylor College of Medicine. Department of Molecular and Human Genetics, and Human Genome Sequencing Center.Houston, TX, USA/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilVIB Center for the Biology of Disease. Leuven, Belgium/ KU Leuven Center for Human Genetics and Leuven Institute for Neurodegenerative Diseases. KULeuven, Belgium/ University of Rome Tor Vergata. Department of Biomedicine and Prevention. Rome, ItalyBaylor College of Medicine. Department of Pediatrics. Section of Pediatric Neurology and Developmental Neuroscience. Houston, TX, USAUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USABaylor College of Medicine. Department of Molecular and Human Genetics, and Human Genome Sequencing Center.Houston, TX, USACenter for Human Genetics. Laboratory for the Genetics of Cognition. KU Leuven, Belgium/ University Hospitals Leuven. Center for Human Genetics. Department of Clinical genetics. Leuven, BelgiumUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAIncreased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup), carrying different duplication sizes, to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition, using multi-electrodes arrays, we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level, we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor, NCH-51, was validated as a potential clinical candidate. Interestingly, this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders